Project Summary A key mechanism for immune evasion and persistent infection by the Lyme disease spirochete, Borrelia burgdorferi, is recombination at the vls locus. To date, the vls locus has not been receptive to direct mutation by any available methods. This fact has created the need for the development of a vls system that is amenable to genetic manipulation. Recently, our lab has generated a mini-vls plasmid construct with high potential to meet this demand. Development of this mini-vls system as a tool for mutational analysis will help bridge a fundamental gap in our understanding of the mechanism behind vlsE antigenic variation. Our long-term goals are to determine the mechanistic aspects of vlsE recombination, and to identify and characterize the unique B. burgdorferi proteins responsible for VlsE antigenic variation. The objective of this application is to identify the mechanistic determinants required for vls switching, and to establish the importance of the cis organization of the vls locus for efficient recombination. The central hypothesis is that that G-run DNA regions directly flanking the vlsE variable cassette, and the 51bp inverted repeat of the vls locus, act as mechanistic DNA elements that are essential for vlsE switching. Furthermore, we hypothesize that antigenic variation requires a cis organization of the locus. The rationale for the proposed research is that identifying key mechanistic elements and important structural components will ultimately provide critical clues to the proteins involved in vls recombination that is critical for persistent infection by the Lyme disease pathogen. Thus, the proposed research is relevant to that part of NIH?s mission that pertains to developing fundamental knowledge that will potentially help to reduce the burdens of human illness and disability. Guided by preliminary data and cited work by other groups, this hypothesis will be tested by pursuing two specific aims: 1) Identify the mechanistic determinants required for vls recombination; and 2) Establish the importance of the cis organization of the vls locus for efficient vlsE recombination. Under the first aim, mutations of the DNA inverted repeat and G-run regions within the vls locus will be generated and cloned into an lp28-1-deficient strain of B. burgdorferi using a unique mini-vls plasmid, and transformants will be used to infect both immunologically-competent and -deficient mice. Under the second aim, spirochetes harboring the vlsE gene and silent cassettes on two separate linear DNA molecules will be examined for vls switching after being passaged through a mouse host. The proposed work is innovative for two main reasons: 1) they entail cloning and genetic manipulation of the full vls locus using a unique mini-vls construct, and 2) they involve transformation of ligated DNA products directly into B. burgdorferi. When applied, the results from the proposed studies are expected to allow the targeting of this system in order to significantly reduce the ability of this pathogen to establish a persistent infection in the mammalian host.